JP2019085376A - Cancer peptide vaccine for prevention of cancer, treatment of cancer or prevention of cancer metastasis in dogs - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a cancer peptide vaccine for prevention of cancer, treatment of cancer and prevention of metastasis of cancer in dogs. A peptide consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 27, which can induce cytotoxic T cells that bind to a canine DLA-88 antigen and target canine cancer cells. .. [Selection diagram] None

Description

  The present invention relates to a peptide effective for preventing cancer, treating cancer or preventing metastasis of cancer in dogs. The present invention further relates to a cancer preventive agent, a cancer therapeutic agent and a cancer metastasis preventive agent comprising the above-mentioned peptide as an active ingredient.

  Of the cancerous malignancies in the dog, oral melanoma is strongly invasive and has a poor prognosis, and in this type of tumor, surgical removal per mandible (or maxilla) results in a significant loss of QOL. There is a tendency to rely on chemotherapy. However, even if it can be temporarily reduced by first-line treatment, it may recur or metastasize, and most of the recurrent cancers are often resistant to the same treatment.

  The cancer peptide vaccine is antigen-presented to dendritic cells in the skin by vaccinating the skin with a synthetic peptide of the same amino acid (AA) sequence as the epitope presented on MHC class I of cancer cells. Is one of the treatments expected to induce cancer-specific cytotoxic T lymphocytes (CTL) and eliminate cancer cells (Fig. 1). The prevention of cancer and the like by a cancer peptide vaccine is reported in Patent Document 1 and Patent Document 2. Although there are experimental reports using mice and numerous case reports in humans, there is no report on implementation in dogs because there is little MHC class I information in dogs.

International Publication No. WO 2015/005479 International Publication No. WO 2015/050259

  The present invention aims to provide a cancer peptide vaccine for preventing cancer, treating cancer or preventing metastasis of cancer in dogs.

  The present inventors have developed canine DLA-88 compatible antigen epitope detection technology with reference to mouse and human MHC class I compatible antigen epitope search technology. In this case, melan-A and TRP-2, which are proteins highly expressed in canine melanoma, and survivin (survivin) expressed in many canine tumors were used as antigens. The DLA-88 type information in dogs is unclear, so the peptide design refers to human HLA type, and 6 types of synthetic peptides for melan-A and survivin among canine cancer-related antigens, TRP For -2, 12 synthetic peptides were prepared. The effects of these synthetic peptides were verified by induction of specific CTL.

  After separation into CD8 + T cells and plate adherent cells from canine peripheral blood mononuclear cells (PBMCs) using magnetic bead antibodies, add canine recombinant GM-CSF / IL-4 and the above synthetic peptide to the plate adherent cells. It was induced to differentiate into antigen presenting cells (APC). After purifying genomic DNA from a sample in which activity to activate CD8 + T cells was observed, the exon 2-3 region of DLA-88 was sequenced and typing was performed. In this way, peptides specific for DLA-88 type for melan-A, survivin and TRP-2 were identified.

  A conventional vaccine combines a full length melan-A protein as an antigen, or a full length melan-A gene as a DNA vaccine, or a mixture of multiple peptides (epitopes), multiple peptides (epitopes) It was a method that was expected to be presented on the DLA-88 type of the dog by chance after being vaccinated using a polypeptide-like substance and taken up into dendritic cells. In this case, when an immune response is not induced, it is assumed that various immune responses (antibody production, CTL response, Th response, immunosuppression, etc.) are simultaneously induced. On the other hand, the present invention is characterized by clarifying the relationship between DLA-88 type and its CTL epitope which had been relied upon by chance, and after DLA-88 type examination, CTLs were pinpointed using pinpointing peptides. It is expected to induce.

That is, the present invention is as follows.
[1] A peptide capable of inducing CTL that targets canine cancer cells by binding to canine DLA-88 antigen consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 4 to 27.
[2] The peptide of [1], which is capable of binding to a specific DLA-88 type and inducing a CTL that targets canine cancer cells having the DLA-88 type.
[3] The cancer may be lymphoma, nasal cavity adenocarcinoma, melanoma (malignant melanoma), malignant histiocytoma, breast adenocarcinoma, osteosarcoma, non-small cell lung cancer, squamous cell carcinoma, bladder urothelial carcinoma, hemangiosarcoma, and The peptide of [1] or [2] selected from the group consisting of mastocythoma.
[4] A cancer peptide vaccine for preventing cancer in a dog, treating cancer or preventing metastasis of cancer, comprising the peptide of any of [1] to [3].
[5] A process of determining DLA-88 of an affected dog, a peptide that DLA-88 can present is selected from peptides consisting of amino acid sequences selected from the group consisting of SEQ ID NOS: 4 to 27 A method of preventing cancer, treating cancer or preventing cancer metastasis in a dog comprising the steps of: and administering a selected peptide.
[6] The cancer may be lymphoma, nasal cavity adenocarcinoma, melanoma (malignant melanoma), malignant histiocytoma, breast adenocarcinoma, osteosarcoma, non-small cell lung cancer, squamous cell carcinoma, bladder urothelial carcinoma, hemangiosarcoma, and The method of [5] selected from the group consisting of mastocytosis.

  The present invention is in the point of finding a combination of an antigen which is a key component of a canine cancer peptide vaccine and a DLA-88 type capable of presenting the antigenic peptide. Cancer peptide vaccines can induce CTL against various malignancies (especially melanomas) in dogs to eliminate the malignancy. Based on the present invention, it is possible to determine DLA-88 type information of a cancer-affected dog and select and provide a vaccine containing a cancer antigen peptide compatible therewith, and the immunotherapy adapted for each dog individual It is possible to apply

  The advantage of cancer peptide vaccine is that unlike the activated lymphocyte therapy and dendritic cell therapy, there is no need for culture equipment, and it is easy to vaccinate the skin and spread to veterinarians in general medical care. It is expected to

It is a figure which shows the outline of a cancer peptide vaccine. FIG. 2 shows the mechanism by which proteins are degraded by the proteasome and recognized as cancer-associated antigens. FIG. 5 shows the presentation of cancer-associated antigens in tumor cells. It is a figure which shows a series of processes of screening of the peptide activity of a cancer peptide vaccine. FIG. 5 shows an example of the results of screening for melan-A peptide with CTL induction.

  Hereinafter, the present invention will be described in detail.

  The peptide that can be used as the cancer peptide vaccine of the present invention is a partial peptide of melan-A, survivin (survivin) or TRP-2 which are canine cancer-related antigens. Here, the cancer-associated antigen refers to an antigen which is expressed not only in cancer cells but also in normal cells, but whose expression is higher in cancer cells than in normal cells. The peptide that can be used as the cancer peptide vaccine of the present invention is a peptide consisting of 9 to 10, preferably 9 amino acids, which is antigen-presented on antigen presenting cells (APC) such as dendritic cells.

  The full-length amino acid sequence of dog melan-A is shown in SEQ ID NO: 1, the full-length amino acid sequence of dog survivin in SEQ ID NO: 2, and the full-length amino acid sequence of dog TRP-2 in SEQ ID NO: 3. The peptide that can be used as the cancer peptide vaccine of the present invention is a partial peptide of a dog melan-A protein consisting of the amino acid sequence shown in SEQ ID NO: 1, and is 9 to 10, preferably 9 consecutive amino acids A partial peptide consisting of a sequence, a partial peptide of a dog survivin protein consisting of an amino acid sequence represented by SEQ ID NO: 2 and a partial peptide consisting of 9 to 10, preferably 9 consecutive amino acid sequences, or SEQ ID NO: It is a partial peptide of TRP-2 protein of the dog which consists of an amino acid sequence represented by 3, and is a partial peptide which consists of 9-10, preferably 9 continuous amino acid sequences.

  In addition, a peptide that can be used as the cancer peptide vaccine of the present invention can bind to a canine MHC (Dog Leukocyte Antigen) class I molecule. That is, it has the same amino acid sequence as the epitope presented on canine MHC class I molecules. Dog MHC class I molecules include DLA-88 molecules.

  The peptide that can be used as the cancer peptide vaccine of the present invention binds to canine MHC class I molecules in the endoplasmic reticulum to form a complex, and antigen presenting cells such as dendritic cells as an MHC class I molecule-peptide complex Carried to the surface and presented. As a result, CTLs are induced by activation of CD8 positive T cells that recognize the antigen, and target cancer cells presenting MHC class I molecule-peptide complexes are targeted. That is, the peptide of the present invention acts as a cancer antigen peptide and can be used for cancer immunotherapy as a cancer vaccine.

  There are many different types (alleles) of DLA-88, and the types of DLA-88 differ in the peptides that can be bound. That is, the immunogenicity of the peptide is confined to the DLA-88 type of affected dogs that seeks to prevent, treat, or prevent the spread of cancer. If a peptide is restricted to the specific DLA-88 type of the affected dog, the peptide is said to be specific DLA-88 type restricted. Therefore, when the peptide of the present invention is administered to dogs as a cancer peptide vaccine, the DLA-88 type of the affected dog to be administered is determined in advance, and the combination of DLA-88 type and the peptide is determined. A peptide that can be presented by DLA-88 of the formula may be administered as a peptide vaccine. Specifically, after collecting canine peripheral blood from cancer-affected dogs and separating it from peripheral blood mononuclear cells (PBMCs) into CD8 + T cells and plate adherent cells using magnetic bead antibodies, the canine recombinant cells can be used as plate adherent cells. GM-CSF / IL-4 and a synthetic peptide are added to induce differentiation into antigen presenting cells (APC). It is determined by mixing culture of purified CD8 + T cells and APC whose proliferation activity has been eliminated by X-ray irradiation and observing the presence or absence of activation of CD8 + T cells over time to determine whether it is induced to CTL. Just do it. By determining the DLA-88 type of the affected dog in which the CTL inducibility is recognized by DLA typing, it is possible to determine the combination of the peptide used and the DLA-88 type to which the peptide can bind. For DLA-88 typing, for example, the description in Barth et al. PLOS ONE, November 28, 2016 doi. Org / 10. 1371 / journal. Pone.

  The determination of DLA-88 can be performed by DNA typing of DLA-88 (DLA-88 typing). Examples of DNA typing include DNA sequencing, PCR (Polymerase Chain Reaction), AFLP (Amplified Fragment Length Polymorphism), SSCP (Single Strand Conformation Polymorphism), and a method using an ASO (Allele Specific Oligonucleotide) probe method. For example, a DNA containing the DLA-88 region may be amplified and sequenced using a primer that amplifies the DLA-88 region. At this time, it is desirable to simultaneously amplify the DLA-12 region as DLA-88 type may enter the other dog's MHC class I antigen, DLA-12 region.

  Specific peptides that can be used as the cancer peptide vaccine of the present invention include the following peptides for the cane melan-A partial peptide, the canine survivin partial peptide, and the canine TRP-2 partial peptide.

Partial peptide of dog melan-A
MPREEAHFI (ml-01) (SEQ ID NO: 4),
LLIACWYCR (ml-41) (SEQ ID NO: 5),
LRGRCPHEG (ml-70) (SEQ ID NO: 6),
FQESNCELV (ml-88) (SEQ ID NO: 7),
LVVPNAPPA (ml-95) (SEQ ID NO: 8), and
AEQSSPPYL (ml-109) (SEQ ID NO: 9)

Partial peptide of dog survivin
MGAPTLPPA (sv-01) (SEQ ID NO: 10),
WQPFLKDHR (sv-10) (SEQ ID NO: 11),
ISTFKNWPF (sv-19) (SEQ ID NO: 12),
FCF KELEGW (sv-59) (SEQ ID NO: 13),
SVKKQFEEL (sv-58) (SEQ ID NO: 14), and
KKQFEELTLG (sv-90) (SEQ ID NO: 15)

Partial peptide of TRP-2 in dogs
AMSPLGWGL (trp-1) (SEQ ID NO: 16),
MAKECCPPL (trp-2) (SEQ ID NO: 17),
WTGHDCNQR (trp-3) (SEQ ID NO: 18),
LERDLQQLI (trp-4) (SEQ ID NO: 19),
ARQDDPTLI (trp-5) (SEQ ID NO: 20),
DDYNRRVTL (trp-6) (SEQ ID NO: 21),
KDIQDCLSL (trp-7) (SEQ ID NO: 22),
SQVMSLHNL (trp-8) (SEQ ID NO: 23),
LHNLVHSFL (trp-9) (SEQ ID NO: 24),
FPPVTNEEL (trp-10) (SEQ ID NO: 25),
VLFFLQYRR (trp-11) (SEQ ID NO: 26), and
LMETHLSDR (trp-12) (SEQ ID NO: 27)

  The peptide that can be used as the peptide vaccine of the present invention has 1 to 3, preferably 1 or 2, and more preferably 1 amino acid deleted or substituted in the amino acid sequence represented by SEQ ID NO: 4 to 27 above. Also included are partial peptides (neoantigens) consisting of added amino acid sequences. Such a peptide is a peptide consisting of an amino acid sequence having a sequence identity of 66.7% or more, preferably 77.8% or more, and more preferably 88.9% or more to the amino acid sequences represented by SEQ ID NOs: 4 to 27.

  The peptide that can be used as the cancer peptide vaccine of the present invention can be synthesized by known peptide synthesis technology or can be produced by recombinant DNA technology.

  The combinations of the peptides that can be used as the cancer peptide vaccine of the present invention and the DLA-88 type that can be bound to and presented to each peptide are shown below. There is a high affinity DLA-88 type detected frequently for each peptide and a low affinity DLA-88 type detected less frequently, with the high affinity DLA-88 type below And low affinity DLA-88 type.

Vacan peptide of Melan-A
MPREEAHFI (ml-01):
High affinity DLA-88
DLA-88 * 003: 02, * 508: 01, * 029: 01, * nov25, * 012: 01, * 028: 01
Low affinity DLA-88
DLA-88 * 017: 01, * 501: 01, * 005: 01, * 051: 01, * nov24
LLIACWYCR (ml-41):
High affinity DLA-88
DLA-88 * 003: 02, * 017: 01, * 501: 01, * 029: 01
Low affinity DLA-88
DLA-88 * 012: 01, * 028: 01, * nov8, * 502: 01, * nov17, * 004: 02, * 032: 01, * nov6
LRGRCPHEG (ml-70):
High affinity DLA-88
DLA-88 * 003: 02, * 017: 01, * 501: 01, * 508: 01
Low affinity DLA-88
DLA-88 * 006: 01, * nov25, * 001: 03, * 005: 01, * nov15, * nov17
FQESNCELV (ml-88):
High affinity DLA-88
DLA-88 * 003: 02, * 017: 01, * 501: 01, * 508: 01, * 029: 01, * nov15
Low affinity DLA-88
DLA-88 * 006: 01, * nov25
LVVPNAPPA (ml-95):
High affinity DLA-88
DLA-88 * 501: 01
Low affinity DLA-88
DLA-88 * 003: 02, * 017: 01, * 508: 01, * 006: 01, * nov9
AEQSSPPYL (ml-109):
High affinity DLA-88
DLA-88 * 003: 02, * 017: 01, * 501: 01, * 508: 01,
Low affinity DLA-88
DLA-88 * 006: 01, * 001: 03, * nov8, * 502: 01

survivin vaccine peptide,
MGAPTLPPA (sv-01):
High affinity DLA-88
DLA-88 * 501: 01, * 003: 02, * 017: 01, * 012: 01, * 029: 01, * 028: 01, * nov37
Low affinity DLA-88
DLA-88 * 508: 01, * 006: 01, * 004: 02, * nov3, * 045: 01, * 038: 01
WQPFLKDHR (sv-10):
High affinity DLA-88
DLA-88 * 501: 01, * 034: 01, * 006: 01, * 012: 01, * 029: 01, * 028: 01, * 004: 02, * nov3
Low affinity DLA-88
DLA-88 * 508: 01, * 502: 01, * 012: 02
ISTFKNWPF (sv-19):
High affinity DLA-88
DLA-88 * 501: 01, * 508: 01, * 034: 01, * 006: 01, * nov25
Low affinity DLA-88
DLA-88 * 002: 01
FCFKELEGW (sv-59):
High affinity DLA-88
DLA-88 * 501: 01, * 508: 01, * 034: 01, * 003: 02, * 017: 01, * 029: 01, * 004: 02
Low affinity DLA-88
DLA-88 * 012: 01, * 028: 01
SVKKQFEEL (sv-88):
High affinity DLA-88
DLA-88 * 501: 01, * 508: 01, * 034: 01, * 029: 01, * 028: 01, * 045: 01
Low affinity DLA-88
DLA-88 * 003: 02, * 017: 01, * nov25, * nov18
KKQFEELTLG (sv-90):
High affinity DLA-88
DLA-88 * 501: 01, * 508: 01, * 034: 01, * 003: 02, * 017: 01, * 006: 01, * 012: 01, * 502: 01,
Low affinity DLA-88
DLA-88 * nov25, * 001: 03, * nov4

TRP-2 vaccine peptide
AMSPLGWGL (trp-1):
High affinity DLA-88
DLA-88 * 051: 01
Low affinity DLA-88
DLA-88 * 006: 01, * 034: 01
MAKECCPPL (trp-2):
High affinity DLA-88
DLA-88 * 006: 01
Low affinity DLA-88
DLA-88 * 034: 01, * 045: 01
WTGHDCNQR (trp-3):
High affinity DLA-88
DLA-88 * 003: 02
Low affinity DLA-88
DLA-88 * 029: 01, * 028: 01, * 001: 03, * nov8, * 012: 01, * 017: 01
LERDLQQLI (trp-4):
High affinity DLA-88
DLA-88 * 004: 02, * 508: 01, * 051: 01
Low affinity DLA-88
DLA-88 * 012: 01, * 005: 01, * 501: 01
ARQDDPTLI (trp-5):
High affinity DLA-88
DLA-88 * 051: 01, * 001: 03
Low affinity DLA-88
DLA-88 * 004: 02, * 029: 01, * 508: 01, * 028: 01, * 034: 01
DDYNRRVTL (trp-6):
High affinity DLA-88
DLA-88 * 004: 02, * 508: 01, * 501: 01
Low affinity DLA-88
DLA-88 * 029: 01, * 051: 01, * 006: 01, * 012: 01
KDIQDCLSL (trp-7):
High affinity DLA-88
DLA-88 * 004: 02
Low affinity DLA-88
DLA-88 * 029: 01, * 001: 03, * nov8
SQVMSLHNL (trp-8):
High affinity DLA-88
DLA-88 * 051: 01
Low affinity DLA-88
DLA-88 * 029: 01, * 508: 01
LHNLVHSFL (trp-9):
High affinity DLA-88
DLA-88 * 004: 02, * 051: 01
Low affinity DLA-88
DLA-88 * 029: 01, * 028: 01, * nov8, * 039: 01
FPPVTNEEL (trp-10):
High affinity DLA-88
DLA-88 * 508: 01
Low affinity DLA-88
DLA-88 * 004: 02, * nov8
VLFFLQYRR (trp-11):
High affinity DLA-88
DLA-88 * 508: 01, * 006: 01
Low affinity DLA-88
DLA-88 * 004: 02, * 029: 01, * 051: 01, * 028: 01, * 003: 02, * 017: 01, * 005: 01
LMETHLSDR (trp-12):
High affinity DLA-88
DLA-88 * 004: 02, * 029: 01, * 508: 01, * 028: 01, * 039: 01
Low affinity DLA-88
DLA 88 * 006: 01, * 001: 03, * 0 12: 01, * 003: 02, * 0 17: 01, * nove 19

  Each peptide can induce CTL and injure cancer cells in the individual dog by administering it to an affected dog having type DLA-88 capable of presenting the peptide. In addition, higher effects can be obtained when administered to affected dogs having high affinity type DLA-88. For example, when MPREEAHFI (ml-01), which is a vaccine peptide of Melan-A, is used to treat cancer in dogs, DLA-88 of affected dogs is DLA-88 * 003: 02, * 017: 01, * 501: 01, * 508: 01, * 029: 01, * nov25, * 005: 01, * 012: 01, * 028: 01, * 051: 01, or * nov24, so the diseased dog in advance It is necessary to confirm that the DLA-88 type of is of these types.

  The cancer peptide vaccine of the present invention can be administered to dogs for the prevention of cancer, treatment of cancer or prevention of metastasis of cancer. In particular, it is suitable for surgical removal of tumor and prevention of metastasis of cancer after radiation therapy. Here, the prevention of cancer means prevention of the onset of cancer, the treatment of cancer means the treatment of cancer of an affected dog, and the prevention of metastasis of cancer means in the blood of cancer cells. When infiltrating, or infiltrating lymph nodes through lymphatic vessels, it means preventing cancer cells from metastasize to other tissues and organs by attacking the cancer cells.

  The cancer targeted by the cancer peptide vaccine of the present invention is not limited, but it expresses melan-A, TRP-2 or survivin, preferably lymphoma, nasal adenocarcinoma, melanoma (malignant melanoma), malignant Histology, breast adenocarcinoma, osteosarcoma, non-small cell lung cancer, squamous cell carcinoma, bladder urothelial carcinoma, hemangiosarcoma, and mastocytoma.

  Among them, the peptide vaccine which is a partial peptide of melan-A is suitable for melanoma, the peptide vaccine which is a partial peptide of TRP-2 is suitable for melanoma, and the peptide vaccine which is a partial peptide of survivin is a lymphoma Suitable for nasal cavity adenocarcinoma, melanoma (malignant melanoma), malignant histiocytoma, breast adenocarcinoma, osteosarcoma, non-small cell lung cancer, squamous cell carcinoma, bladder urothelial carcinoma, hemangiosarcoma and mastocytoma.

  Although the dose of the peptide vaccine of the present invention can be changed depending on the type of cancer, severity and the like, it is 10 μg to 100 μg, preferably about 50 μg at a time. The administration may be performed only once a week after the initial administration, and may be administered once every several weeks to several months thereafter for maintenance thereof. The peptide vaccine of the present invention may be in the form of a sterile aqueous or liposoluble solution or emulsion. Furthermore, salts, buffers, immune enhancers such as adjuvants, carriers, etc. may be included. The peptide vaccine may be administered parenterally, such as intradermal, intravenous, intranasal, intramuscular, etc., or orally. Among these, intradermal administration is preferred.

  One of the above peptides may be administered, or a plurality of peptides may be administered in combination. For example, two, three, four, five, six, seven, eight, nine, ten, eleven, twenty-four peptides consisting of the amino acid sequences shown in SEQ ID NOs: 4 to 27. Twelve, thirteen, fourteen, fifteen, sixteen, seventeen, eighteen, nineteen, twenty, twenty-one, twenty-two, twenty-three, twenty-three or twenty-four may be administered in combination. The invention also encompasses preparations or kits combining multiple peptides.

  Also, at this time, peptides different in the DLA-88 type to be presented may be combined. This combination can provide a combination preparation of peptides effective for many affected dogs, regardless of the DLA-88 type.

  The cancer peptide vaccine of the present invention may be administered together with an adjuvant. As the adjuvant, Freund's incomplete adjuvant, cell components such as mycobacteria, gram negative bacteria and gram positive bacteria, and known adjuvants such as bacterial and virus-derived CpG-ODN can be used. In addition, the vaccine of the present invention may be taken by animals such as dogs while contained in drinking water or feed. The invention also encompasses drinking water and feed containing vaccines.

The DNA encoding the cancer peptide vaccine of the present invention or a vector containing the DNA can also be used to prevent cancer, treat cancer or prevent metastasis of cancer. As a vector, viral vectors such as adenovirus, adeno-associated virus, retrovirus, detoxified retrovirus, and vaccinia virus can be used. The DNA encoding the cancer peptide vaccine of the present invention may be introduced into these viral vectors, and the viral vectors may be infected into affected dogs. The administration method is preferably intramuscular injection. In addition, an expression vector such as a plasmid vector can be used to introduce a target DNA into cells or tissues in a culture system of PBMC after blood collection from a diseased dog. For example, genes can be introduced into cells by the lipofection method, phosphate-calcium coprecipitation method, DEAE-dextran method, direct injection of DNA using a micro glass tube, or the like. The DNA encoding the cancer peptide vaccine of the present invention may be operatively introduced into a vector, and at this time, a promoter, enhancer, poly A signal or the like for transcribing DNA may be included. The dosage of DNA and vector can be appropriately determined according to the type of cancer and the severity of cancer, but for example, when using a vector, 10 ⁷ to 10 ⁹ pfu (plaque forming unit) of vector can be administered Good. When the DNA encoding the cancer peptide vaccine of the present invention or a vector containing the DNA is administered to an affected dog, the peptide is expressed in vivo, binds to the dog's MHC class I molecule in the endoplasmic reticulum, and the complex is They are formed and transported to the surface of antigen presenting cells as MHC class I molecule-peptide complexes and presented. As a result, antigen-specific immune responses induce and enhance CTL, and target and attack cancer cells presenting MHC class I molecule-peptide complexes.

  Furthermore, the peptide, DNA and vector of the present invention can be used in combination with an agent that releases the effect of suppressing the antitumor immune function. Such agents include immune checkpoint inhibitors, and examples include PD-1 / PD-L1 inhibitors such as anti-PD-1 antibodies and anti-PD-L1 antibodies, and anti-CTLA4 antibodies.

  The present invention includes a method of preventing cancer, treating cancer, and preventing metastasis of cancer by administering the above-mentioned peptide, or DNA or vector to an affected dog. At this time, DLA-88 type of the affected dog may be determined before administration, and a peptide that can be presented by DLA-88 type may be selected and administered. That is, the present invention comprises the steps of determining type DLA-88 of an affected dog, selecting a peptide that type DLA-88 can present, and administering a selected peptide. Includes prevention, treatment of cancer, and methods to prevent cancer metastasis.

  The invention encompasses methods of inducing CTL in vitro using the peptides of the invention, administering the CTL to a dog, preventing cancer, treating cancer, and preventing metastasis of cancer. .

  That is, the method comprises culturing the cancer peptide vaccine of the present invention with peripheral blood monocytes collected from a diseased dog, and inducing and producing a peptide-presenting antigen-presenting cell. Further, the present invention includes a method of inducing and producing CTL, which are cultured with peripheral blood lymphocytes collected from an affected dog, to specifically injure cancer cells presenting the peptide. Specifically, the peptide, the antigen-presenting cell, and the CD8 + T cell may be co-cultured. Furthermore, ex vivo methods are also included, wherein the antigen-presenting cells or CTL thus produced are administered to affected dogs to prevent cancer, treat cancer and prevent metastasis of cancer.

  The present invention is specifically described by the following examples, but the present invention is not limited by these examples.

Materials and methods
1. Dog peripheral blood mononuclear cells (PBMC)
The remaining amount (subject to disposal) after blood tests of the affected dogs at the Animal Health Center at the Institute of Veterinary Medicine, Japan was used. The utilization of blood from affected dogs (affected dogs) has been approved by the Ethics Committee of the Animal Medical Center of the Veterinary Life Sciences University of Japan (H24.5 / 8). PBMC separation from dog blood (200 to 800 μL) was performed after density gradient centrifugation (800 G, 15 to 20 minutes) using a 15 mL centrifuge tube (TrueLine) containing 2 mL of blood separation solution Lymphoprep (STEMCELL). Obtained by recovery of the bulb fraction.

2. Synthetic peptides
Obtain amino acid sequences from dog melan-A (NM_001194968), dog survivin (AY741504), and dog TRP-2 (AB766380) information on GenBank registry, and search sites for cleavage by proteasome activity (http: //www.mpiib -berlin.mpg.de/MAPPP/cleavage.html) and a search site for peptides that can be presented in various MHC molecules (https://www-bimas.cit.nih.gov/molbio/hla_bind/) The predicted peptide sequence was designed for presentation on DLA-88. Table 1 shows melan-A, Table 2 shows survinvin, and Table 3 shows TRP-2. These peptide sequences were synthesized at a purity of 95% or more by a consignment service (Biologica).

3. Purification of CD8 + T cells
The PBMCs are reacted with FITC-labeled anti-dog CD8 antibody (Serotec) for 30 minutes at 4 ° C, washed with phosphate buffer (PBS), and then similarly reacted with Biotin-labeled anti-FITC antibody (BioLegend) for 30 minutes at 4 ° C, Washed with PBS. These cells are reacted with Streptavidin-labeled magnetic bead antibody (BDIMag Particles Plus DM) for 15 minutes at 4 ° C, then made up to 600 μL with RPMI medium, allowed to act on a dedicated magnet stand for 6 minutes, and CD8 + T cells (Purified around 90%) were collected and cell number was adjusted (3-4 x 10 ⁴ / well x 6 well or 12 well).

4. Induction of antigen-presenting cells (APC) (such as dendritic cells)
Depending on the number of cells from which PBMC were obtained, aliquot (10 ⁵ / well) into 6 wells or 12 wells of flat-bottom 96 well plate (Thermo), react with 5% CO ₂ at 37 ° C for 40 to 60 minutes, plate adherent cells (4-5 x 10 ⁴ / well) was prepared. Cells that did not adhere were harvested and cryopreserved after DNA purification. Canine recombinant GM-CSF 5 ng / mL (PrimeGene), canine recombinant IL-4 5 ng / mL (Immunotechnology Co.) and various synthetic peptides 10 μg / mL were added to each plate well for 90 to 120 minutes. The cells were induced to differentiate into antigen-presenting cells (APCs) by These APCs lost their proliferative activity by X-ray irradiation of 30 Gy (CP-160 type Faxtron X-ray).

5. Screening of peptide activity APCs from which proliferative activity had been lost and purified CD8 + T cells were mixed and cultured, and 10 days before, the presence or absence of induced CTL induction was determined by microscopic examination over time (FIG. 4). That is, the peripheral blood of dogs can be obtained after examination of surplus from the University Medical Center for Medical Research and separated from its peripheral blood mononuclear cells (PBMC) into CD8 + T cells and plate-adherent cells using magnetic bead antibodies, and then plated. Canine recombinant GM-CSF / IL-4 and synthetic peptides were added to adherent cells to induce differentiation into antigen-presenting cells (APC). The purified CD8 + T cells and the APC whose proliferation activity was lost by X-ray irradiation were mixed and cultured, and the presence or absence of activation of the CD8 + T cells was observed over time. The number of items to be performed was adjusted according to the blood volume of the obtained sample and the number of purified CD8 + T cells.

A procedure of steps 1 to 5 is shown in FIG.
FIG. 5 shows an example of the result of screening for melan-A peptide having CTL induction.

6. DLA-88 inspection
Although there are only a few types of DLA-12, cases in which DLA-88 enters in the DLA-12 region were detected. Therefore, a primer that simultaneously amplifies only the DLA-88 or DLA-12 region gave a PCR product of 5.6 to 5.7 kb including the entire region of the gene (all exons, introns and untranslated regions from the promoter region). Then, using DLA-88 specific primer and DLA-12 specific primer, 1.6 kb including polymorphism rich exon 2 to exon 3 was amplified by PCR method using PCR product as a template. The nucleotide sequence of the resulting PCR product was decoded by Sanger method.

  When it is difficult to decipher the base sequence or the presence of a new allele is considered, the PCR product was subcloned and the clone was deciphered by the Sanger method. For the determination of the DLA allele, the base determined using the sequence analysis software Assign (Conexio) or Sequencher ver. 5.0.1 (Gene Codes) with the DLA-88 or DLA-12 allele released as a reference sequence Comparative analysis with the sequences was performed to determine the DLA-88 and DLA-12 alleles possessed by each sample.

result
Identification of Melan-A Peptide-Displayed DLA Type Up to the present, 692 samples were analyzed, and 57 samples of Melan-A were found to be capable of inducing CTL against any of 6 peptides. As a result of performing typing of DLA-88 type | mold of the sample with these activities, the table | surface which described the activity of DLA-88 type allele and six types of peptides was completed (Table 4). The DLA-88 detected at a frequency of 3 times or more has a high affinity of 003: 02, 017: 01, 501: 01, 028: 01, 029: 01, 508: 01, nov15 ^* , nov25 ^* 8. The species and DLA-88 detected twice have low affinity as 001: 03, 004: 02, 005: 01, 006: 01, 012: 01, 032: 01, 051: 01, 502: 01, 13 types of nov6 ^* , nov8 ^* , nov9 ^* , nov17 ^* and nov24 ^* were found.

  As shown in Table 4, DLA-88 capable of antigen-presenting Melan-A peptide ml-01, ml-41, ml-70, ml-88, ml-95, and ml-109 could be identified. Taking the view of this table as an example, DLA-88 * 003: 02 type, DLA-88 * 017: 01 type, DLA-88 * 501: 01 type is a type that can present all these six peptides. As can be appreciated, six peptides can be used as cancer vaccines for these types of dogs.

  On the other hand, since DLA-88 * 508: 01 can present ml-01, ml-70, ml-88, ml-95, and ml-109 other than ml-41 as antigens, five peptides are used as cancer vaccines. It can be used.

  Similarly, ml-70, ml-88, ml-95, ml-109 for the DLA-88 * 006: 01 type, ml-01, ml-41, ml- for the DLA-88 * 029: 01 type. A peptide adapted to each DLA-88 type can be used as a cancer vaccine, such as 88 for DLA-88 * 001: 03 type and ml-70 for ml-109 (hereinafter omitted).

Identification of Survivin Peptide-presenting DLA Type Up to the present, 692 samples were analyzed, and it was found that 81 samples of Survivin have CTL inducibility against any of 6 peptides. As a result of performing typing of DLA-88 type | mold of the sample with these activities, the table | surface which described the activity of DLA-88 type allele and 6 types of peptides was completed (Table 5). The DLA-88 detected at a frequency of three or more times has high affinity as 003: 02, 004: 02, 006: 01, 012: 01, 017: 01, 028: 01, 029: 01, 034: 01 , 501: 01, 502: 01, 508: 01, nov 25 ^* , 13 nov 37 ^* , and twice detected, DLA-88 has low affinity as 001: 03, 012: 02, 038: 01, Seven types of 045: 01, nov3 ^* , nov4 ^* and nov18 ^* were found.

  As shown in Table 5, DLA-88 capable of antigen-presenting survivin peptides sv-01, sv-10, sv-19, sv-59, sv-88 and sv-90 could be identified. Taking the view of this table as an example, it can be seen that DLA-88 * 501: 01 and DLA-88 * 508: 01 are types capable of presenting all six types of peptides, and in these types of dogs In contrast, six peptides can be used as cancer vaccines.

  On the other hand, since DLA-88 * 034: 01 can present sv-10, sv-19, sv-59, sv-88 and sv-90 other than sv-01 as antigens, five peptides are used as cancer vaccines. It can be used. Similarly, the DLA-88 * 003: 02 type and the DLA-88 * 017: 01 type are sv-01, sv-59, sv-88, sv-90, and the DLA-88 * 006: 01 type is sv -01, sv-10, sv-19, sv-90, DLA-88 * 012: 01 for the type sv-01, sv-10, sv-59, sv-90, DLA-88 * 028: 01 Type and DLA-88 * 029: 01 for type sv-01, sv-10, sv-59, sv-88, DLA-88 * 004: 02 for type sv-01, sv-10, sv-59 , DLA-88 * nov25 for sv-19, sv-88, sv-90, DLA-88 * 502: 01 for type SV-10, sv-90 (abbreviated below) and so on. A peptide adapted to -88 type can be used as a cancer vaccine.

Identification of TRP-2 peptide-displayed DLA type: Up to the present, 692 samples were analyzed, and it was found that TRP-2 has CTL inducibility for any of 12 peptides in 66 samples. As a result of performing typing of DLA-88 type | mold of the sample with these activities, the table | surface which described the activity of DLA-88 type allele and 12 types of peptides was completed (Table 6). The DLA-88 detected at a frequency of three or more times has high affinity as 001: 03, 003: 02, 004: 02, 006: 01, 017: 01, 028: 01, 029: 01, 039: 01 , 051: 01, 501: 01, 508: 01, 12 types of nov 8 ^* and DLA-88 detected twice have 005: 01, 012: 01, 034: 01, 045: 01 as low affinity. , Five species of nov 19 ^* ( ^* ; new will be clarified by phylogenetic tree analysis). As shown in Table 6, DLA-88 type capable of antigen-presenting TRP-2 peptide trp-1 to trp-12 could be identified. Taking the view of this table as an example, DLA-88 * 004: 02 and DLA-88 * 029: 01 can present 8 of these 12 peptides, and DLA-88 * 004: 02 can trp-4, trp-5, trp-6, trp-7, trp-9, trp-10, trp-11, trp-12, DLA-88 * 029: 01 type is trp-3, trp-5, trp-6, trp-7, trp-8, trp-9, trp-11, trp-12 can be used as cancer vaccines.

  On the other hand, DLA-88 * 508: 01 type is trp-4, trp-5, trp-6, trp-8, trp-10, trp-11, trp-12, DLA-88 * 051: 01 is trp Seven types of peptides can be used as cancer vaccines because -1, trp-4, trp-5, trp-6, trp-8, trp-9, and trp-11 can be antigen-presented. Similarly, the DLA-88 * 006: 01 type is trp-1, trp-2, trp-6, trp-11, trp-12, and the DLA-88 * 028: 01 type is trp-3, trp-5, trp-9, trp-11, trp-12, DLA-88 * 001: 03 is trp-3, trp-5, trp-7, trp-12, DLA-88 * 012: 01 is trp- 3, trp-4, trp-6, trp-12, DLA-88 * nov8 type trp-3, trp-7, trp-9, trp-10 (abbreviated below) and so on. Type-adapted peptides can be used as cancer vaccines.

  Thus, the present invention relates to the combination of a specific amino acid sequence of a cancer antigen Melan-A, survivin, TRP-2 with a peptide based on information on canine DLA-88 to induce cancer antigen specific CTL. It was established in an in vitro system using canine PBMC. In performing immunotherapy for canine melanoma or adenocarcinoma or the like to which this is applied, a peptide of a specific amino acid sequence that conforms to the type can be used as a cancer vaccine after examination of type DLA-88 in advance.

  According to conventional studies, it has been suggested from the past studies that similar in vitro response can be obtained with high probability in the canine cancer immune response, and van der Bruggen et al. The amino acid sequence MAGE-1 (MZ2-E) capable of inducing CTL using patient's blood was presented, and a new immunotherapy was proposed (Science. 1991 254 (5038): 1643-1647.). Subsequently, Rosenberg SA et al. Actually vaccinated melanoma patients using synthetic peptides and reported clinical data of cases where effects were seen (Nat Med. 1998 4 (3): 321-327.). Many other human peptide vaccine cases have been practiced to date.

  The peptide of the present invention can be used as a cancer peptide vaccine for treating cancer in dogs, preventing cancer and preventing metastasis of cancer.

SEQ ID NOs: 4 to 27

Claims (6)

  A peptide comprising an amino acid sequence selected from the group consisting of SEQ ID NOS: 4-27, which is capable of binding to the canine DLA-88 antigen to induce cytotoxic T cells targeting canine cancer cells.   The peptide according to claim 1, which is capable of inducing a cytotoxic T cell that binds to a specific DLA-88 type and targets canine cancer cells having the DLA-88 type.   Cancer from lymphoma, nasal cavity adenocarcinoma, melanoma (malignant melanoma), malignant histiocytoma, breast adenocarcinoma, osteosarcoma, non-small cell lung cancer, squamous cell carcinoma, bladder transitional cell carcinoma, hemangiosarcoma, and mast cell tumor The peptide according to claim 1 or 2, selected from the group consisting of   A cancer peptide vaccine for preventing cancer in a dog, treating cancer or preventing metastasis of cancer, comprising the peptide according to any one of claims 1 to 3.   Determining the type DLA-88 of the affected dog, selecting among the peptides consisting of the amino acid sequences selected from the group consisting of SEQ ID NO: 4-27, the peptide that can be presented by the type DLA-88, and A method of preventing cancer, treating cancer or preventing metastasis of cancer in a dog comprising administering a selected peptide.   The cancer may be lymphoma, nasal cavity adenocarcinoma, melanoma (malignant melanoma), malignant histiocytoma, breast adenocarcinoma, osteosarcoma, non-small cell lung cancer, squamous cell carcinoma, bladder urothelial carcinoma, hemangiosarcoma, and mast cell tumor A method according to claim 5, selected from the group consisting of

Images